Wnt proteins are lipid-modified, short-range signals that control stem cell self-renewal and tissue regeneration. We identified a population of Wnt responsive cells in the pulp cavity, characterized their function, and then created a pulp injury. The repair response was evaluated over time using molecular, cellular, and quantitative assays. We tested how healing was impacted by wound environments in which Wnt signaling was amplified. We found that a Wnt-amplified environment was associated with superior pulp healing. Although cell death was still rampant, the number of cells undergoing apoptosis was significantly reduced. This resulted in significantly better survival of injured pulp cells, and resulted in the formation of more tertiary dentin. We engineered a liposomereconstituted form of WNT3A then tested whether this biomimetic compound could activate cells in the injured tooth pulp and stimulate dentin regeneration. Pulp cells responded to the elevated Wnt stimulus by differentiating into secretory odontoblasts. Thus, transiently amplifying the body's natural Wnt response resulted in improved pulp vitality. These data have direct clinical implications for treating dental caries, the most prevalent disease affecting mankind.

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Functional tissue engineering for bone augmentation requires the appropriate combination of biomaterials, mesenchymal stem cells, and specific differentiation factors. Therefore, we investigated the morphology, attachment, viability, and proliferation of human dental pulp stem cells cultured in xeno-free conditions in human serum medium seeded on β-tricalcium phosphate/poly(l-lactic acid/caprolactone) three-dimensional biomaterial scaffold. Additionally, osteogenic inducers dexamethasone and vitamin D3 were compared to achieve osteogenic differentiation. Dental pulp stem cells cultured in human serum medium maintained their morphology; furthermore, cells attached, remained viable, and increased in cell number within the scaffold. Alkaline phosphatase staining showed the osteogenic potential of dental pulp stem cells under the influence of osteogenic medium containing vitamin D3 or dexamethasone within the scaffolds. Maintenance of dental pulp stem cells for 14 days in osteogenic medium containing vitamin D3 resulted in significant increase in osteogenic markers as shown at mRNA level in comparison to osteogenic medium containing dexamethasone. The results of this study show that osteogenic medium containing vitamin D3 osteo-induced dental pulp stem cells cultured in human serum medium within β-tricalcium phosphate/poly(l-lactic acid/caprolactone) three-dimensional biomaterial, which could be directly translated clinically.

Introduction:Dental pulp stem cells (DPSCs) are an accessible cell source with therapeutic applicability in regeneration of damaged tissues. Current techniques for expansion of DPSCs require the use of Fetal Bovine Serum (FBS). However, animal-derived reagents stage safety issues in clinical therapy. By expanding DPSCs in serumfree/ xenofree medium (SF/XF-M) or in medium containing human serum (HS-M), the problems can be eliminated. Therefore, the aim of our study was to identify suitable cell culture media alternatives for DPSCs. Methods:We studied the isolation, proliferation, morphology, cell surface markers (CD29, CD44, CD90, CD105, CD31, CD45 and CD146), stemness markers expression (Oct3/4, Sox2, Nanog and SSEA-4) and in vitro multilineage differentiation of DPSCs in HS-M or SF/XF-M in comparison to FBS-M.Results: DPSCs expressed the cell surface and stemness markers in all studied conditions. The proliferation analysis of cells cultured in different HS concentrations revealed that cells isolated in 20% HS-M and passaged in 10% or 15% HS-M supported the cell growth. Direct isolation of cells in SF/XF-M did not support cell proliferation. Therefore, cells cultured in 20% HS-M were used for further SF/XF-M studies. However, proliferation of DPSCs was significantly lower in SF/XF-M when compared with cells cultured in FBS-M and HS-M. In addition, proliferation of DPSCs in SF/XF-M could be enhanced by addition of 1% HS in cell culture medium. There were differences in osteogenic, chondrogenic and adipogenic differentiation efficacy between cells cultured in FBS, HS and SF/XF differentation media. More pronounced adipogenic and osteogenic differentiation was observed in HS differentiation medium, however, in FBS-M cultured cells more effective chondrogenic differentiation was detected. Conclusions:Our results indicate that HS is a suitable alternative to FBS for the expansion of DPSCs. The composition of SF/XF-M needs to be further optimized in terms of cell expandability and differentiation efficiency to reach clinical applicability.